Method of modifying cytotoxic cells and uses thereof

ABSTRACT

TALL-104 cells, and other cytotoxic T cell lines, may be modified to increase the cytotoxicity thereof, to enhance growth properties, and/or to provide a preferred phenotype, e.g., expression of cell surface antigens, function, e.g., change in cytokine production profile, by culturing the cells in an effective amount of IL-15, optionally followed by gamma irradiation to halt proliferation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a 371 of PCT/US00/04548, filed Feb. 23, 2000 which claims thebenefit of the priority of U.S. patent application Ser. No. 60/121,482,filed Feb. 24, 1999.

FIELD OF THE INVENTION

The invention relates generally to the modification of cytotoxic T cellsby treatment with a selected cytokine, and to the use of such modifiedcells in cancer therapy.

BACKGROUND OF THE INVENTION

The human T cell line TALL-104 (CD3/TCRαβ*CD8*CD16⁻) [A. Cesano et al,In Vitro Cell. Dev. Biol., 28A:648 (1992); A. Cesano et al, J. Immunol.,151:2943-2957 (1993); and A. Cesano et al, Cancer Immunol, Immunoth.,40:139 (1995)] is endowed with MHC non-restricted killer activity andhas been reported as useful, when lethally irradiated, against a broadrange of tumors across several species, while sparing cells from normaltissues. As taught by the inventors' prior publications and patentscited above, unmodified TALL-104 cells are available from the AmericanType Culture Collection, 10801 University Boulevard Manassas, Va.20110-2209 under Accession Number CRL 11386 and are described in U.S.Pat. No. 5,272,082. These cells may be preferably modified by lethalγ-irradiation and/or by stimulation in the cytokine interleukin 2 (IL-2)or Interleukin 12 (IL-12) to provide them with an increased cytotoxicityagainst tumor and virus-infected targets.

Such modification methods have been described in detail in InternationalPatent Publication No. WO94/26284, published Nov. 24, 1994, which isincorporated by reference herein. For example, one modification stepincludes in vitro treatment of the TALL-104 cells with one or both ofthe two interleukins, recombinant human (rh) IL-2 and rhIL-12. When usedindependently to treat the cell line, IL-2 and IL-12 can induce the cellline's cytotoxic activity. When these cytokines are used together tomodify the cell line, the modified cell line displays additive orincreased cytotoxic effects. This results in a significant increase incytotoxic activity and recycling capability, ultimately leading to 100%elimination of tumor targets at an E:T ratio<0.1:1 [Cesano et al, J.Immunol., 151:2943 (1993)].

Another known modification step involves the exposure of the TALL-104cell line to lethal irradiation to confer irreversible loss of growthcapability with full retention of cytotoxic activity, both in vitro andin vivo. This is achieved by subjecting the cell line to y-irradiationjust prior to its use. Preferably, the cells are irradiated at 4000 radsusing a ¹³⁷Cs source. As described in International Patent PublicationNo. WO94/26284, irradiation of TALL-104 cells provides a modifiedcytotoxic cell line that has lost its proliferative ability and,therefore, the possibility of growing in an unrestrained fashion in therag, recipient organism. These modified TALL-104 cells have been used inmethods for the treatment of various cancers in humans and animals. See,also, U.S. Pat. Nos. 5,683,690; 5,702,702 and 5,820,856, andInternational patent publication No. WO98/48630, all incorporated hereinby reference.

Other cytotoxic cells have also been described, such as the TALL-103/2cells. See, U.S. Pat. No. 5,272,082 and A. Cesano et al, J. Immunol,151:2943-2957 (1993); S. Visonneau et al, Cell Immunol., 165:252-265(1995); and A. Cesano et al, J. Immunol., 160:1106-1115 (1998). However,TALL-103/2 cells, stimulated with IL-2 or IL-12, have been noted to havea limited spectrum of tumor target reactivities and display low levelsof killing. These cells do not grow in severe combined immuno-deficient(SCID) mice. Thus, at present, TALL-103/2 cells have not appearedpromising for clinical use.

Among the known cytokines, Interleukin-15 (IL-15) is a relatively novelT cell growth factor that shares some activities and receptor componentswith IL-2 [U.S. Pat. No. 5,747,024; J. G. Giri et al, J. Leuko. Biol.,57(5):763-6 (May 1995); L. S. Quinn et al, Endocrinol., 136(8):3669-72(August 1995)]. IL-15 utilizes the β and γ chains of the IL-2 receptorfor signal transduction, but uses a different subunit (α) to bind to thecells. The expression pattern of IL-15 α receptor is distinct from thatof IL-2 α receptor. IL-15 has been shown to induce LAK cell functions invitro at high doses of about 100 ng/ml by a CD18-dependent,perforin-related mechanism [A. M. Gamero et al, Cancer Res.,55(21):4988-94 (November 1995)]. IL-15 is produced by monocytes anddendritic cells and has been shown to induce cytokine production inhuman T helper cells, and adhesion receptor redistribution in Tlymphocytes. It has been described to stimulate proliferation of γδ Tcells and act synergistically with other stimuli in inducing lymphokineproduction thereby [See, also, W. E. Carson et al, J. Clin. Invest.96(6):2578-82 (December 1995); H. Jonuleit et al, J. Immunol.,158(6):2610-5 (Mar. 15, 1997); V. E. Garcia et al, J. Immunol.,160(9):4322-9 (May 1998); A. Mori et al, J. Immunol 156(7):2400-5 (April1996); M. Nieto et al, Euro. J. Immunol., 26(6):1302-7 (June 1996); M.K. Kennedy et al, J. Clin. Immunol., 16(3):134-43 (May 1996)]. IL-15 hasalso been described as a vaccine adjuvant [U.S. Pat. No. 5,747,0241], atherapeutic [U.S. Pat. No. 5,660,824], and an inducer of angiogenesis[A. L. Angiolillo et al, Biochem. Biophys. Res. Comm., 2(1):231-7 (Apr.7, 1997)]. IL-15 has been said to have IL-2-like stimulating activitieson T lymphocytes and NK cells [P. Allavena et al, J. Leuko. Biol.,61(6):729-35 (June 1997); J. P. DiSanto, Current Biol., 7(7):R424-6(Jul. 1, 1997); R. Evans et al., Cell. Immunol., 179(1):66-73 (Jul. 10,1997)].

There exists a need in the art for methods for further enhancing thecharacteristics of cytotoxic T cells useful for therapy.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of modifying, orreversibly modifying, the phenotype and function of cytotoxic T cellswhile retaining the cytotoxicity of the cells comprising the steps of:

(a) culturing said cells in an effective amount of IL-15 therebyobtaining a high yield of a cell having a first phenotype;

(b) culturing the IL-15 stimulated cells in an effective amount of IL-2,thereby altering the first phenotype to a second phenotype; and

(c) optionally repeating steps (a) and (b) a selected number of times.

In another aspect, the invention provides a method of modifying acytotoxic T cells while retaining the cytotoxicity of the cellscomprising the steps of:

(a) culturing said cells in an effective amount of IL-2, therebyobtaining a first modified cell;

(b) culturing the IL-2 stimulated cells in an effective amount of IL-15;thereby obtaining a second modified cell; and

(c) optionally repeating steps (a) and (b) a selected number of times.

The first and second modified cells from either method above demonstratea change in at least one characteristic, such as increasedproliferation, differentiation, growth, phenotype, adhesion moleculeexpression, biodistribution, cytokine production profile, level ofcytotoxic activity, and tumor target spectrum. Desirably the cells areTALL-104 cells or TALL-103/2 cells.

In one embodiment of the first method, TALL-104 cells are cultured in aneffective amount of IL-15, wherein said cells grow at a rate faster thanwhen stimulated by IL-2, and have an altered phenotypic profile; andthen the IL-15 stimulated TALL-104 cells are cultured in an effectiveamount of IL-2. In an embodiment of the second embodiment, themodification of cell characteristics is accomplished by first culturingTALL-104 cells in an effective amount of IL-2 and then culturing theIL-2 stimulated TALL-104 cells in an effective amount of IL-15.

In yet another aspect, the invention provides a method of modifyingTALL-104 cells comprising culturing TALL-104 cells in an effectiveamount of IL-15, wherein said cells grow at a rate faster than whenstimulated by IL-2, and have an altered phenotypic, cytotoxic andcytokine profile. The modified cells have an increased level ofcytotoxicity or another change in a characteristic such as increasedproliferation, differentiation, growth, phenotype, adhesion moleculeexpression, biodistribution, cytokine production profile, and tumortarget spectrum. In one embodiment of this method the cytokine profileincludes increased expression of IL-10, GM-CSF, TNF-α and TNF-5 anddecreased expression of gamma interferon (IFN-γ) by the modifiedTALL-104 cells. In another embodiment, the modified phenotype includesincreased expression of the cytotoxic adhesion/activation marker CD56and/or decreased expression of the adhesion molecule CD38.

In still another aspect, the invention provides a method for increasingthe levels of cytotoxic activity and spectrum of tumor targetrecognition of TALL-103/2 cells comprising culturing TALL-103/2 cells inan effective amount of IL-15, wherein said cells grow at a faster rateand have an expanded tumor target spectrum of cytotoxicity than whenstimulated by IL-2.

In yet a further aspect, the invention provides modified TALL-104 cells,which are produced by stimulating said cells in an effective amount ofIL-15.

In another aspect, the invention provides modified TALL-103/2 cellshaving an increased cytotoxicity, which are produced by stimulating saidcells in an effective amount of IL-15.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph which illustrates that IL-15 supports greater TALL-104cell proliferation in vitro. The symbol □ indicates the 5 ng/ml dosageof IL-15; the symbol ⋄ indicates the 100 U/ml dosage of IL-2. Growth ofcells is measured by a metabolic surrogate marker, lactate (mg/dl), overdays in culture.

FIG. 2 is a bar graph illustrating that upon expansion of TALL-104 cellsin vivo in SCID mice and re-adaptation to tissue culture conditions,IL-15 induces quicker differentiation of TALL-104 cells into cytotoxiccells in comparison to the effects of IL-2. Undifferentiated TALL-104cells, extracted from SCID mouse spleens, were cultured for one weekwith either IL-2 or IL-15, and then tested for cytotoxicity againsteither K562 or Raji tumor cells. Cytotoxicity is demonstrated by percentlysis of the tumor cells. The open bar indicates IL-2 treatment; thestrippled bar indicates IL-15 treatment. TALL-104 cells stimulated inIL-15 also show higher levels of cytotoxic molecules such as perforin,serine esterases (SE) and TIA-1, an apoptosis inducing molecule.

FIG. 3 is a graph indicating that IL-15 induces higher expression ofcytotoxic adhesion/activation marker CD56, both as percent of positivecells in the total TALL-104 population and at the single cell level, asthe number of molecules present on each cell (higher density). Thesymbol □ indicates IL-2; the symbol ⋄ indicates IL-15. Results areplotted as % CD56+cells over time (days) in culture.

FIG. 4 is a graph demonstrating that undifferentiated TALL-104 cellsfreshly obtained from the SCID mouse have a higher expression of CD2 andthat with time in culture CD2 surface levels decline. However, thisdecline is slower in TALL-104 cells grown in IL-15 than in IL-2. Thesymbol 0 indicates IL-2; the symbol 0 indicates IL-15. Results areplotted as % CD2+cells over time (days) in culture.

FIG. 5 is a graph demonstrating that TALL-104 cells grown in IL-15 havelower expression of the adhesion molecule CD38. The symbol □ indicatesIL-2; the symbol ⋄ indicates IL-15. Results are plotted as % CD38+cellsover time (days) in culture.

FIG. 6 is a bar graph illustrating the comparative effects of IL-15 andIL-2 on the induction of GM-CSF from TALL-104 cells. On the X axis arethe stimuli used to trigger GM-CSF production. The graph shows thatTALL-104 cells grown in IL-15 have a baseline level of GM-CSFproduction, and respond to OKT3 monoclonal antibody with higherproduction of GM-CSF than cells grown in IL-2.

FIG. 7 is a bar graph illustrating the comparative effects of IL-15 andIL-2 on the induction of IL-10 from TALL-104 cells. On the X axis arethe stimuli used to trigger IL-10 production. The graph shows thatTALL-104 cells grown in IL-15 have a baseline level of IL-10 production,and respond to OKT3 monoclonal antibody with higher production of IL-10than cells grown in IL-2.

FIG. 8 is a bar graph illustrating the comparative effects of IL-15 andIL-2 on the induction of TNF-α from TALL-104 cells. On the X axis arethe stimuli used to trigger TNF-α production. The graph shows thatTALL-104 cells grown in IL-15 have a baseline level of TNF-α production,and respond to OKT3 monoclonal antibody with higher production of TNF-αthan cells grown in IL-2.

FIG. 9 is a bar graph illustrating the comparative effects of IL-15 andIL-2 on the induction of TNF-β from TALL-104 cells. On the X axis arethe stimuli used to trigger TNF-β production. The graph shows thatTALL-104 cells grown in IL-15 have a baseline level of TNF-β production,and respond to OKT3 monoclonal antibody with higher production of TNF-βthan cells grown in IL-2.

FIG. 10 is a bar graph illustrating the comparative effects of IL-15 andIL-2 on the induction of IFN-γ from TALL-104 cells. On the X axis arethe stimuli used to trigger IFN-γ production. The graph shows thatTALL-104 cells grown in IL-15 have a baseline level of IFN-γ production,and respond to OKT3 monoclonal antibody with lower production of IFN-γthan cells grown in IL-2.

FIG. 11 is a graph which illustrates that IL-15 supports the growth ofTALL-103/2 cells in culture. The symbol □ indicates the dosage of IL-2in U/ml; the symbol ⋄ indicates the dosage of IL-15 in ng/ml. Growth ofcells is indicated by cpm in ³H-TdR proliferation assays. The X-axisshows the concentration of the cytokines.

FIG. 12 is a bar graph demonstrating that IL-15 (strippled bars)supports the cytotoxic phenotype of TALL-103/2 cells and broadens thespectrum of target recognition by these cells. The cells were culturedfor a week in either IL-15 or IL-2 (clear bars), and then exposed toK562 tumor cells (sensitive target) and H160 or Daudi tumor cells(resistant targets). Cytotoxicity is measured by % lysis of the targetcells in ⁵¹Cr release assays.

FIG. 13 is a bar graph which illustrates the comparative effect of IL-2and IL-15 on the induction of IFNγ in TALL-103/2 cells. The X axis showsthe cytokine dosage. Results show that IL-2 is a better inducer of thiscytokine.

FIG. 14 is a bar graph which illustrates the comparative effect of IL-2and IL-15 on the induction of TNF-βin TALL-103/2 cells. The X axis showscytokine dosage. IL-15 induces higher levels of TNF-β at theconcentration of 10 μg/ml.

FIG. 15 is a bar graph which illustrates the comparative effect of IL-2and IL-15 on the induction of TNF-α in TALL-103/2 cells. Similar levelsof TNF-α are induced by the two cytokines.

FIG. 16 is a bar graph which illustrates the comparative effect of IL-2and IL-15 on the induction of IL-10 in TALL-103/2 cells. IL-15 is abetter inducer of IL-10 as compared to IL-2.

DETAILED DESCRIPTION OF THE INVENTION

Cytotoxic T cell lines, such as TALL-104, have found use in clinicalsettings, such as the treatment of cancers, when administered in vivo,or when employed in ex vivo therapeutic regimens. Still other cytotoxicT cell lines, such as TALL-103/2 could be clinically useful if theirtarget specificity was broadened and their growth in culture improved.The inventors have now discovered novel methods for increasing thecytotoxicity of these cells, altering their phenotypes and spectrum oftarget recognition, and, increasing their yield in culture.

Such modifications can be introduced to TALL-104 or TALL-103/2 cells,and are anticipated to be introduced to other cytotoxic T cells bystimulating the cells in IL-15, rather than, or in addition to, IL-2.Such IL-15 stimulation may optionally be followed by exposing thestimulated TALL-104 cell line to lethal irradiation to conferirreversible loss of growth capability with retention of cytotoxicactivity, both in vitro and in vivo. This may be achieved by subjectingthe cell line to γ-irradiation just prior to its use. Preferably, thecells are irradiated at 4000 rads using a ¹³⁷Cs source, similar to theprocess described in International Patent Publication No. WO94/26284.Such irradiation of the IL-15 stimulated TALL-104 cells provides amodified cytotoxic cell line that has lost its proliferative abilityand, therefore, the possibility of growing in an unrestrained fashion inthe recipient organism.

Thus, in one embodiment, TALL-104 cells are prepared as follows.TALL-104 cells (ATCC CRL 11386) are exponentially grown in tissueculture in the presence of recombinant human (rh) IL-15. The resultingproliferation of the cytokine stimulated TALL-104 cells (as measured by³H-TdR uptake) is higher at plateau doses of IL-15 than at plateau dosesof IL-2. A “plateau dose” is the dose at which maximal activity isreached, e.g., the optimal dose (see FIG. 1). These cells alsodemonstrate increased ability to adhere to plastic in vitro (andpotentially to endothelium in vivo) by increase in expression ofadhesion molecules. These modified TALL-104 cells also demonstrateincreased cytotoxic function, as shown by higher levels of killing,increased spectrum of tumor target recognition, and a quicker and moreeffective kinetic of induction of lytic proteins, such as PFP, SE1 andSE2, and TIA1. Cells grown in an optimal dose of IL-15 generally showhigher levels of cytotoxic activity, as compared to the same cells in anoptimal dose of IL-2 (see FIG. 2, which demonstrates a significantincrease in cytotoxicity against NK-sensitive K562 cells andNK-resistant Raji cells, as compared to the same TALL-104 cellsstimulated in IL-2). The cells also demonstrate an increased expressionof the cytotoxic adhesion marker CD56 (FIG. 3). TALL-104 cells grown inIL-15 have higher baseline levels of cytokines and respond to stimuli,such as antibodies and target cells, producing higher levels ofcytokines than TALL-110 grown in IL-2, with the exception of gammainterferon (IFN-γ), which is produced in higher levels by stimulation ofTALL-104 cells in IL-2. The same results were obtained with TALL-103/2cells.

Therefore, according to one embodiment of this invention, TALL-104 cellsmay be grown in IL-15 simply to increase the yield thereof, and thengrown in IL-2 to reproduce the IL-2 cytotoxic phenotype previously usedin clinical therapies for cancer. Alternatively, one may grow TALL-104cells in IL-15 and use the IL-15 phenotype where enhanced adhesion toendothelium is desired in some clinical applications. The inventors havedetermined that one may reversibly switch the IL-15 and IL-2 phenotypesof TALL-104 by sequential growth of the cells in one and then the otherof these two cytokines, as desired. The biodistribution of the TALL-110cells may also be affected differently by the two cytokines, based onthe different levels of expression of adhesion molecules.

In yet another embodiment of this invention, TALL-103/2 cells may alsobe modified by stimulation in IL-15. In this instance, the TALL-103/2cells will grow more rapidly in culture (FIG. 11) when stimulated withIL-15. Most significantly, when TALL 103/2 cells are stimulated inIL-15, their target recognition expands, and these cells may then beused against more tumor cell types. For example, FIG. 12 shows theresults of stimulation of TALL 103/2 cells with IL-15 vs. IL-2. TheIL-15 TALL 103/2 cells are able to recognize and kill HL60 and Daudicells, against which the IL-2 stimulated TALL 103/2 cells were notcytotoxic. Additionally, the stimulation of the TALL 103/2 cells withIL-15 alters the cytokine production by the cells. See, for example,FIGS. 12-16, which showing cytokines that are likely to be involved inthe anti-tumor activity of the killer cells. Thus, changing the cytokineprofile can result in clinical changes, both in terms of efficacy and/ortoxicity.

Based on the effects that IL-15 has on these two cytotoxic T cell lines,it is anticipated that similar effects may be obtained with othercytotoxic T cell lines. Thus, IL-15 may be employed in a method forreversibly altering the phenotype of cytotoxic T cells by culturing saidcells in IL-15, thereby obtaining a high yield of a cell having a firstphenotype; followed by culturing these cells in IL-2, thereby alteringthe first phenotype to a second phenotype. The second phenotype may bereturned to the first phenotype by further culturing in IL-15 again, ifdesired. The IL-15 phenotypes are characterized by enhanced growthkinetics, increased cytotoxicity, enhanced cytokine production, and,likely, increased adhesion to vasculature.

These IL-15 stimulated cytotoxic T cells may then be employed in methodsfor in vivo and ex vivo therapy of cancer, and for other uses for whichTALL-104 cells are known, as described in the US patents incorporated byreference above. These modified cells may also be employed as researchreagents, as reagents for screening the effect of proposed developmentaldrugs on their cytotoxic activity, as reagents for the study of theirexpression of adhesion molecules or cell surface markers, as well as forthe production of cytokines or other biological molecules expressed bythe modified cells.

The following examples demonstrate the effect of IL-15 on TALL-104 cellsand TALL-103/2 cells. These examples in illustrate the preferred methodsof the invention. These examples are illustrative only and do not limitthe scope of the invention.

EXAMPLE 1 Growth of Tall-104 Cells in IL-15

TALL-104 cells were grown in endotoxin-free Iscove's modified Dulbecco'smedium (Gibco-BRL, Grand Island, N.Y.) supplemented with 10%heat-inactivated fetal bovine serum (Sigma) and 100 U/ml rhIL-2 (ChironTherapeutics, Emeryville, Calif.) or rh IL-15 (1-5 μg/ml) (R& D Systems)in a humidified incubator at 37° C. with 10% CO₂ in tissue culture fortwo weeks.

The cells were then examined for differences in characteristics such asgrowth, phenotype, cytokine profile, and cytotoxicity, biodistributionand tumor target spectrum of the cultures, and the results were reportedin FIGS. 1-10.

A. Proliferation

As shown in FIG. 1, TALL-104 cells grown in the plateau dose (5 ng/ml)of IL-15 proliferate in vitro faster than do the same cells grown in theplateau dose 100 U/ml) of IL-2.

B. Target Spectrum

In another experiment, the IL-2- or IL-15-treated TALL-104 cells wereexpanded in vivo in SCID mice and re-adapted to tissue cultureconditions. IL-15 induced quicker differentiation of TALL-104 cells intocytotoxic cells in comparison to the effects of IL-2. UndifferentiatedTALL-104 cells, extracted from SCID mouse spleens, were cultured for oneweek with either IL-2 or IL-15, and then tested for cytotoxicity againsteither K562 or Raji tumor cells. The IL-2-treated TALL-104 cells wereonly marginally (≦10%) cytotoxic, as demonstrated by percent lysis ofthe tumor cells, for K562 cells. In contrast, the TALL-104 cellsstimulated with IL-15 lysed about 40% more K562 tumor cells. TheIL-2-treated cells lysed no Raji cells, whereas the IL-15 treatedTALL-104 cells lysed almost 60% of these cells. See FIG. 2. The IL-15treated cells also showed higher levels of cytotoxic molecules, such asperforin, serine esterases (SE) and TIA-1, an apoptosis inducingmolecule.

C. Phenotype

TALL-104 cells treated with IL-15 express higher levels of thecytotoxic/adhesion/activation marker CD56, both as percent of positivecells in the total TALL-104 population and at the single cell level, asthe number of molecules present on each cell (higher density), than doTALL-104 cells treated with IL-2. See FIG. 3.

Undifferentiated TALL-104 cells freshly obtained from the SCID mice havea high expression of CD2. With time in culture, the CD2 surface levelsdecline. However, this decline when compared for TALL-104 cells grown inIL-2 or IL-15 as described above, was demonstrated to be slower in theIL-15 stimulated cells. See FIG. 4.

TALL-104 cells grown in IL-15 were also shown to have lower expressionof the adhesion molecule CD38 than TALL-104 cells grown in IL-2. SeeFIG. 5.

D. Cytokine Profile

The TALL-103 cells, grown in either IL-2 or IL-15 as above, werestimulated to trigger cytokine production with OKT3 (anti-CD3), Moon-1(anti-CD31), or IB4 (anti-CD36) monoclonal antibodies or by exposure toK562 cells.

As seen in FIG. 6, TALL-104 cells grown in IL-15 have a baseline levelof GM-CSF production, and respond to OKT3 monoclonal antibody with asignificantly higher production of GM-CSF than cells grown in IL-2.

As seen in FIG. 7, TALL-104 cells grown in IL-15 have a baseline levelof IL-10 production, and respond to OKT3 monoclonal antibody withsignificantly higher production of IL-10 than cells grown in IL-2.

As seen in FIG. 8, TALL-104 cells grown in IL-15 have a baseline levelof TNF-α production, and respond to OKT3 monoclonal antibody with higherproduction of TNF-α than cells grown in IL-2.

As seen in FIG. 9, TALL-104 cells grown in IL-15 have a baseline levelof TNF-β production, and respond to OKT3 monoclonal antibody with higherproduction of TNF-α than cells grown in IL-2.

As seen in FIG. 10, TALL-104 cells grown in IL-15 have a baseline levelof IFN-γ production, and respond to OKT3 monoclonal antibody with lowerproduction of IFN-γ than cells grown in IL-2.

EXAMPLE 2 Growth of Tall-103/2 Cells in IL-15

TALL-103/2 cells were grown in endotoxin-free Iscove's modifiedDulbecco's medium (Gibco-BRL, Grand Island, N.Y.) supplemented with 10%heat-inactivated fetal bovine serum (Sigma) and 100 U/ml rhIL-2 (ChironTherapeutics, Emeryvile, Calif.) or rh IL-15 (1-5 μg/ml) [R&D Systems]in a humidified incubator at 37° C. with 10% C₂ in tissue culture fortwo weeks. The cells were then examined for differences incharacteristics such as growth, phenotype, cytokine profile, andcytotoxicity, biodistribution and tumor target spectrum of the cultures,and the results were reported in FIGS. 1-10.

A. Phenotype

The surface phenotype of the cultured cells were compared to determinethe effect of the two cytokines. The results are reported below in TableI as % positive cells; the mean fluorescence intensity (on a scale withthe upper limit of 200) is in parentheses and provides an indication ofthe antigen density, i.e., the number of molecules/cell.

TABLE I Cell IL-15 Surface IL-2 Treated Treated Antigen TALL-103/2TALL-103/2 CD3 96.5(92) 35.6(62) CD2 53.5(75) 43.5(61) CD4  4.4(60)15(62) CD8 90.1(131) 78.9(113) CD56 41.6(60) 67.4(123) LFA-3 99.3(125)88(118) ICAM-1 71.5(84) 44.9(81) CD45RO 81.3(83) 72.2(86) CD38 70.5(74)32.5(62) CD31 35.6(61)  2.3(66)

B. Proliferation

In ³H-TdR proliferation assays, TALL-103/2 cells grown in IL-15 showedgreater proliferation than the cells grown in IL-2 at stimulatingcytokine doses greater than 1 ng/ml IL-15. See FIG. 11.

C. Target Spectrum

TALL-103/2 cells were cultured for a week in either IL-15 or IL-2, andthen exposed to K562 tumor cells, HL60 tumor cells or Daudi tumor cells.Cytotoxicity was measured by % lysis of the target cells in ⁵¹Cr releaseassays. As demonstrated in FIG. 12, the IL-15 treated cells caused lysisof all three tumor cell types. The IL-2 treated cells were cytotoxiconly for the K562 cells. Thus, the method of this invention supportedthe cytotoxic phenotype of TALL-103/2 cells and broadened the spectrumof target recognition by these cells.

D. Cytokine Profile

The IL-2-treated and IL-15-treated TALL-103/2 cells were also evaluatedfor dose-dependent cytokine production. As revealed by FIGS. 13-16, theIL-15 induces production of cytokines from the cells, which is differentfrom that produced by stimulating the cells with IL-2.

IL-2 stimulation induces better expression of IFNγ in TALL-103/2 cells,than does IL-15 stimulation (FIG. 13).

IL-15 induces higher levels of TNF-β at the concentration of 10 μg/ml(FIG. 14), than does IL-2 stimulation.

Similar levels of TNF-α are induced by the two cytokines (FIG. 15).

IL-15-stimulated TALL-103/2 cells produce greater amounts of IL-10 atconcentrations over 10 ng/ml IL-15 (FIG. 16).

Numerous modifications and variations of the present invention areincluded in the above-identified specification and are expected to beobvious to one of skill in the art. Such modifications and alterationsto the methods of the present invention are believed to be encompassedin the scope of the claims appended hereto.

What is claimed is:
 1. A method of modifying TALL-104 cells (ATCC CRL11386) comprising: culturing TALL-104 cells in an effective amount ofIL-15, wherein said cells grow at a rate faster than when stimulated byIL-2, and have an altered phenotypic, cytotoxic and cytokine profile. 2.The method according to claim 1 wherein said modified cells have anincreased level of cytotoxicity.
 3. The method according to claim 1,wherein said modified cells demonstrate a change in a characteristicselected from the group consisting of increased proliferation,differentiation, growth, phenotype, adhesion molecule expression,biodistribution, cytokine production profile, and tumor target spectrum.4. The method according to claim 3 wherein said cytokine productionprofile is characterized by increased expression of IL-10, increasedexpression of GM-CSF, increased expression of TNF-α, increasedexpression of TNF-β, and decreased expression of gamma interferon uponstimulation with anti-CD3 antibody.
 5. The method according to claim 3wherein said phenotype is characterized by increased expression of thecytotoxic adhesion/activation marker CD56 and decreased expression ofthe adhesion molecule CD38.